DNA sequencing has helped revolutionize the way that genes and genomes are studied, and has led to a greater understanding of most aspects of biology. Nevertheless, with efforts underway to map and sequence the genomes of a variety of organisms, the need to improve the efficiency of DNA sequencing has never been greater (1). One of the major problems associated with sequencing large segments of DNA is obtaining sequence information beyond the limits of a single primer extension event. Several techniques are currently used to acquire sequences within the interior of a DNA insert; these include: i) the synthesis of custom primers to "walk" along a segment of DNA (2, 3), ii) shotgun subcloning, which requires a high degree of redundancy for complete sequence recovery (4), or iii) the construction of overlapping exonuclease deletion clones (3, 5). Each of these methods is time-consuming, idiosyncratic and therefore difficult to automate, and/or costly.
Alternatively, transposable elements have been adapted for DNA mapping and sequencing. Examples include: .gamma..delta. (6), Tn5 (7), Tn10 (8), as well as derivatives of these and other transposons. Although these approaches generally offer great promise, the insertion step is performed in vivo in E. coli; hence, transposition may occur into either the plasmid target or the E. coli genome, complicating the recovery of target insertions. An additional difficulty arises from host effects on insertion randomness, i.e., "hotspots" and "coldspots" of integration are often observed in vivo (9).
The complete DNA integration reaction employed by certain retroviruses and retrotransposons as part of their normal life cycles can be carried out completely in vitro (10-14) offering a possible alternative to in vivo transposon insertion techniques for DNA sequencing.
There is a need in the art for a simple, reliable technique for generating sets of DNA templates for sequencing any target. In particular there is a need for sets of DNA templates which are amenable to automated sequencing with a single set of primers.